Methods for genotyping by hybridization analysis

Abstract

This invention provides methods for determining the genotype of organisms by hybridization analysis and, more specifically, to establishing the relatedness of individual organisms within a species. The present invention provides addressable arrays, comprising diversity panels of nucleic acid molecules, in which the molecules on the array are addressable or uniquely identifiable in some fashion. A diversity panel is the result of a method that can distinguish sequence differences between nucleic acid samples. As taught herein, a variety of methods may be used to generate diversity panels. Subsequent to the generation of the diversity panel, the nucleic acid products of the diversity panel are separated for application onto an array. The separated diversity panel is then delivered onto a substrate to create an addressable array and hybridized with labeled nucleic acids. The genotype of an organism is determined by the pattern of hybridization.

Description

FIELD OF THE INVENTION [0001] This invention relates generally to determining the genotype of organisms by hybridization analysis and, more specifically, to establishing the relatedness of individual organisms within a species. BACKGROUND OF THE INVENTION [0002] A genotype is the genetic constitution of an individual or group. Variations in genotype are essential for commercial breeding programs, diagnostics, monitoring genetic-based diseases, following spread of pathogens, determining parentage, and the like. While determining the nucleic acid sequence of genomic DNA is one way to unambiguously establish a genotype of an individual, it is not currently practicable to accomplish, especially in organisms with complex genomes. [0003] Genotypes can be more readily described in terms of genetic markers. A genetic marker identifies a specific region or locus in the genome. Thus, the more genetic markers, the finer defined is the genotype. A genetic marker becomes particularly useful when...

AI Technical Summary

Benefits of technology

[0104] Isolation of Polymorphism / Transgenic Plants

[0105] The nucleic acid molecule comprising a detected polymorphism is isolated using techniques known in the art. The nucleic acid molecule may be cloned in an appropriate vector if not already cloned. In turn, the clone may be mapped on the genome using conventional techniques or mapped to a collection of BAC or YAC clones. The nucleotide sequence may be determined as well.

[0106] In certain embodiments, the polymorphic nucleic acid molecule may be used to transform a host cell, either a plant or animal. Methods to make transgenic plants are known in the art. Depending upon the nature of the transgenic sequence it may be desirable to operatively link the sequence to a promoter that are active in plants. Such promoters may be constitutive, such as the 35S CaMV promoter, tissue-dependent, such as those active only in root tissues, stage-dependent, such as those active during embryogenesis, or the like. Examples of promoters are readily found in public databases (e.g., GenBank).

[0107] Following Polymorphisms Through Introgression / Back-Crossing

[0108] Introgression of specific alleles is a goal frequently pursued in plant breeding as well as laboratory animal breeding programs. The end product is a plant or animal nearly identical to the desired parent except for a specific region of the genome that is contributed by another individual. For example, the advent of mice strains with identical backgrounds but differing at the Major Histocompatibility Complex locus was instrumental in understanding the effect of MHC differences on organ transplantation. In crop development, a desirable trait, such as disease resistance, may be identified in a plant, but is generally introgressed into elite varieties that are better suited to the local environment, soil and climate or to consumer preferences than the original plant. The introgression is usually performed by repeated backcrosses of the new individual with the elite parent. During the introgression of the genes that account for the traits, means to follow that trait are necessary. In some cases, the trait may not be assayable in the field except under defined conditions (e.g., challenge with the pathogen). It is advantageous, however, to have a marker for the gene i.e. a polymorphism genetically linked to the desired trait, which can then be assayed to identify suitable plants for the breeding program. In order to accelerate the speed of the introgression process, it is also important to monitor that the rest of the genome is as similar as possible to the elite parent. In that regard, the determination of a genotype encompassing a large number of markers in parallel, provided by the present invention is a distinct advantage. The present invention provides the means to follow specific markers linked to a desirable traits, as well as genome-wide markers measuring the extent of reconversion of the genome, and allows for high throughput screening.

[0109] Constructing a Genetic Map; Discovering Important Genes through Association Studies

Problems solved by technology

While determining the nucleic acid sequence of genomic DNA is one way to unambiguously establish a genotype of an individual, it is not currently practicable to accomplish, especially in organisms with complex genomes.

All of these genotyping methods suffer from the laborious requirement to analyze only a single organism at a time.

A further burden in some of these methods is the need for pre-identification of a polymorphism before analysis of other individuals (U.S. Pat. No. 6,100,030).

Still others of these methods depend upon expensive materials and time-intensive gel electrophoresis, resulting in a low-throughput.

Furthermore, these methods that base identity on size suffer from additional difficulties in precisely correlating bands on gels with alleles.

In this technique however, a genotype of an organism is not established.

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